Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991-03-26 to 1991-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
EEC Directive 84/449 EEC, EEC Publication No. L251, September 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Mai 12, 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US Food and Drug Administration, Bureau of Foods, Toxicological Principles for the Safety Assessment of Direct Food Additives and Colour Additives used in Food (1982)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Calcium hydrogen phosphonate
EC Number:
244-182-1
EC Name:
Calcium hydrogen phosphonate
Cas Number:
21056-98-4
IUPAC Name:
calcium phosphonate
Details on test material:
- Name of test material (as cited in study report): RV 777
- Substance type: Reaction mass of dialuminium tricalcium hexaoxide (CAS No. 12042-78-3, EC No. 234-932-6) and calcium hydrogen phosphonate (CAS No. 21056-98-4, EC No. 244-182-1)
- Analytical purity: 90 %
- Physical state: white solid
- Lot/batch No.: CH. 659 B
- Bulk density: 0.62 g/ml
- Expiration date of the lot/batch: November 22, 1995
- Stability in vehicle: stable in polyethylene glycol for 48 hours
- Storage condition of test material: at room temperature, dry
- Other: stability under storage conditions: stable for 5 years

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: BRL Ltd., Basel Switzerland
- Age at study initiation: approximately 6 weeks
- Housing: Group housing of 5 animals per sex per gage in stainless steel suspended cages with wire mesh floors.
- Diet: standard pelleted laboratory animal diet (Kliba, Klingentalmühle AG, Kaiseraugst, Switzerland) ad libitum
- Water: tap water ad libitum
- Acclimation period: At least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod: Lighting was 12 hours artificial flourescent light and 12 hours dark per day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
TEST SUBSTANCE FORMULATION

Method: The test substance was weighed into a glass flask on an analytical balance and the vehicle (w/w) added. Adjustement was made for specific gravity of vehicle (1.127).
Frequency of test substance formulation: Daily immediately prior to dosing.
Homogeneity of test substance in vehicle: By use of a magnetic stirrer, an electric shaker and a homogeniser. Homogeneity during treatment was maintained using a magnetic stirrer.
Storage instructions for test substance formulation: At ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared were analysed to check homogeneity and accuracy of preparation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day (m/f)
Basis:
other: gavage, dose volume: 5 ml/kg body weight, actual dose volume: calculated weekly according to the latest body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg bw/day) to provide a basis for selection of dose levels for a study of longer duration.
No differences of biological significance were observed in clinical appearence, body weight, food consumption, macroscopic appearance or liver weights between the treated groups.
Based on these observations, a high treatment level of 1000 mg/kg bw/day was selected for a study of 28 days duration.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
--clinical signs: at least once daily
-- mortality/viability: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Severity of observations were graded

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding termination, prior to overnight fasting

FOOD CONSUMPTION: Yes
- Time schedule: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study period, but no quantitative assessment introduced as no effect was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before commencement of treatment and during the last week of treatment
- Both eyes were examined following instillation of tropicamide solution (5 mg/ml)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Yes (overnight before blood sampling, but water was provided)
- How many animals: all rats/sex/group
- Parameters examined: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution, total leucocyte count, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Animals fasted: Yes (overnight before blood sampling, but water was provided)
- How many animals: all rats/sex/group
- Parameters examined: glucose, urea, creatinine, bilirubine, aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), gamma-glutamyl transferase (G-GT), sodium, potassium, chloride, calcium, phosphorus, total protein, albumin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- How many animals: all rats/sex/group

ORGAN WEIGHTS: Yes
- How many animals: all rats/sex/group
- Organs examined: adrenal glands, heart, kidneys, liver, spleen, testes

HISTOPATHOLOGY: Yes
- How many animals: all rats/sex/group
- Tissues and organs samples fixed: adrenal glands, aorta, brain, cecum, cervix, clitoral gland, colon, duodenum, epididymides, esophagus, eyes with optic nerve and Harderian gland, female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), nasopharynx, ovaries, pancreas, pituitary gland, preputial gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions
- Slides of tissues and organs examined: adrenals, heart, kidneys, liver, spleen, stomach
Statistics:
The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- The exact Fisher-test was applied to the ophthalmoscopic examination data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY

There were no clinical signs of toxicity or behavioural changes over the 29 day observation period that were considered to be related to treatment.

Excessive salivation was noted intermittently in treated males and females receiving 50, 200 or 1000 mg/kg bw/day. Since this may be attributed to a possible irritant effect or bad taste of the test substance, it was considered not to represent a clear sign of toxicity.

Incidental findings (i.e. scabs, alopecia and rough coat) as noted in single animals receiving 200 or 1000 mg/kg bw/day, showed no relationship to treatment and were therefore considered to have occurred fortuitously.

MORTALITY

No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN

Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period.

A minor statistically significant difference arising between body weight gain of females receiving 1000 mg/kg bw/day and control females was within normal biological variation for rats of this age and strain and considered to have arisen by chance.

FOOD CONSUMPTION

There were no differences in food consumption before and after allowance for body weight between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION

There were no differences between the pretest examination and the examination at week 4 that could be attributed to treatment with the test substance.

HAEMATOLOGY

Haematological parameters of treated rats were considered not to have been affected by treatment.

The incidentally statistically significantly decreased relative number of monocytes between control females and females receiving 1000 mg/kg bw/day was considered not to have been arisen as a result of toxicity.

CLINICAL CHEMISTRY

There were no differences noted between control and treated rats that could be related to treatment with the test substance.

Statistically significantly increased glucose values between treated and control males were, in the absence of a treatment-related distribution, considered to have occurred fortuitously.

ORGAN WEIGHTS

Organ weights and relative organ weights of treated animals were indistinguishable from those of control animals.

GROSS PATHOLOGY

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as an effect of treatment.

Pelvic dilatation in the kidney, which was noted incidentally among control and treated females, is commonly noted in rats of this age and strain and considered not to represent a sign of toxicity.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no microscopic findings noted that were considered to be treatment related. The small number of changes recorded in treated animals were within the range commonly seen for rats of this age and strain.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No mortality occured during the study. There were no changes in clinical appearance, body weights, food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examinations, organ weights and microscopic examination that were considered to be an effect of treatment up to and including the highest tested dose level of 1000 mg/kg bw/day.
Executive summary:

In a subacute toxicity study according to OECD Guideline No. 407, Mai 12, 1981 and EEC Directive 84/449/EEC, Part B.7, September 1984, RV 777 (90 % a.i.) was administered to groups of 5 Wistar rats per dose and sex by gavage at dose levels of 0, 50, 200, and 1000 mg/kg bw/day.

In this study up to and including the highest dose tested (1000 mg/kg bw/day), no mortality occured and there were no changes in clinical appearance, body weights, food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examinations, organ weights and microscopic examination that were considered to be an effect of treatment.

The NOAEL is 1000 mg/kg bw/day.

This subacute oral toxicity study in the rat is acceptable and satisfies the guideline requirements.